The present invention relates to means and methods for conducting chemiluminescent immunoassay testing and, more particularly, to initiating and monitoring a chemiluminescent reaction in a plurality of such assays, of different types, on a single immunoassay instrument, in a single procedure, using a plurality of analyte-specific, labeled reagents and a triggering reagent combination, wherein the phenomenon referred to as “RLU shift,” in which the luminescence output can increase or decrease, is reduced or eliminated.
Assays are available to test a variety of body functions, including, for example, anemia, growth, thyroid, hypertension, tumor markers, neonatal conditions, the adrenal/pituitary system, bone and mineral metabolism, and the reproductive system. Immunoassay analyzers, such as chemiluminescent immunoassay analyzers, which automatically assay specimens, are one such device.
Chemiluminescence is a chemical reaction that emits energy in the form of light. The intensity of the luminescence when compared to a control intensity provides a measure of the concentration of the sample being tested. For example, during a chemiluminescent assay, a test sample and an analyte-specific, labeled reagent, such as an acridinium ester antigen or an acridinium ester antibody, are dispensed in a cuvette. Next, a solid phase reagent, such as paramagnetic particles having a binding substance coupled thereto, is added to the sample-containing cuvette. The sample-containing cuvette is then incubated.
During incubation, acridinium ester-labeled antibodies bind specifically to the antigen of interest in the sample. Alternatively, acridinium ester-labeled antigens compete with the antigen or interest already in the sample, to bind to the available antibody in the sample. Unbound labeled reagent is washed away.
Chemical reagents, often referred to as “trigger” reagents, are then added to the sample-containing cuvette to oxidize the analyte-specific, labeled reagent, or, more specifically, to initiate the chemiluminescent reaction. The trigger reagents, conventionally, include an acid reagent and a base reagent. First, an acid reagent, e.g., hydrogen peroxide, is dispensed into the sample-containing cuvette, to initiate oxidation. Then a base reagent is dispensed into the sample-containing cuvette, to alter the state of the environment from acidic to basic, which accelerates oxidation.
After the chemiluminescent reaction has been initiated, oxidation of the label to its excited state and its subsequent return to the ground state generates a flash, which typically lasts a few seconds. Conventionally, this flash is detected by a system-integrated luminometer. By examining the luminescence output resulting from mixing the test sample and trigger reagents, the automated analyzer can determine the concentration of the specific chemical constituent, for which the testing is being performed, in the patient's specimen.
More specifically, the luminescence output of the chemiluminescent reaction, i.e., the intensity of the flash, which is measured and expressed in relative light units (RLU), can be compared to a calibrated test standard, to determine the amount, for example, of bound, labeled antibody or antigen in the patient's blood.
Addition of a surfactant, such as a cationic detergent, to the base reagent is common. Advantageously, addition of a surfactant increases the intensity of light. However, when a surfactant is added, analyzers become susceptible to a phenomenon referred to as “RLU shift”. During an “RLU shift”, the luminescence output of the test sample can increase or decrease, which is to say “shift”, by as much as fifteen or more percent. As a result, the measurements of specific chemical constituent concentration can be over- or under-estimated. This causes unacceptable total imprecision.
Therefore, it would be advantageous to be able to perform a plurality of such assays, of different types, on a single immunoassay instrument, in a single procedure, using a plurality of labels, and triggering reagents to initiate a chemiluminescent reaction for each of the labels sequentially.